Compounds and methods for treating nematode infections

ABSTRACT

The present application relates to the treatment of nematode infections. For example, the application relates to the use of compounds of Formula (I) and/or (II) as defined herein for treatment of a nematode infection or a disease, disorder or condition arising from a nematode infection:

RELATED APPLICATIONS

The present application is a continuation-in-part of co-pending U.S.patent application Ser. No. 16/565,917 filed on Sep. 10, 2019, whichclaims the benefit of priority of U.S. provisional patent applicationNo. 62/730,286 filed on Sep. 12, 2018, the contents of each of which areincorporated herein by reference in their entirety.

FIELD

The present application relates to the treatment of nematode infections.For example, the application relates to the use of compounds of Formula(I), and/or Formula (II) as defined herein for treatment of a nematodeinfection or a disease, disorder or condition arising from a nematodeinfection.

INTRODUCTION

The burden of parasitic nematodes on humanity is severe. The WHOestimates that over two billion people are infected with at least oneparasitic nematode species. Chronic infection can cause dietarydeficiency, anemia, developmental delay, elephantiasis, blindness, anddeath. Human infection in the west is increasing, coincident with awarming climate and the movement of sub-tropical species northward.Intestinal nematode infections alone are responsible for an estimateddisease burden of 3.4 million disability-adjusted life-years.Furthermore, nematode infestation of food increases costs andcontributes to malnutrition. Nematodes have evolved widespreadresistance to nearly every anthelmintic (anti-worm) drug on the market.Hence, there is a dire need for the development of new compounds thatkill parasitic worms.

Most anthelmintics disrupt the worm's nervous system, which ultimatelyallows the mammalian host to clear the infection. For example, keytargets of ivermectin are glutamate-gated chloride channels of thenervous system. Ivermectin agonizes these channels and hyperpolarizesthe cell, in turn leading to paralysis. Similarly, levamisole agonizesnicotinic acetylcholine receptors leading to depolarization of musclesand, in turn, paralysis.

SUMMARY

It has been shown herein that compounds of Formula (I) and/or (II)provide inhibitory activity against species of nematodes such asCooperia oncophora, Haemonchus contortus, and Caenorhabditis elegans.

Accordingly, in one embodiment, the present application includes methodsand uses for treating or preventing a nematode infection comprisingadministering an effective amount of a compound of Formula (I)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereofwhereinL is C₀₋₄ alkylene;

R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; and

R² is H, NO₂, Cl, F or CN.

In one embodiment, the present application includes methods and uses fortreating or preventing a nematode infection comprising administering aneffective amount of a compound of Formula (II)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereof.

In some embodiments, the present application includes methods and usesof treating or preventing a disease, disorder or condition arising froma nematode infection comprising administering an effective amount acompound of Formula (I)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereofwhereinL is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN.

In some embodiments, the present application includes methods and usesof treating or preventing a disease, disorder or condition arising froma nematode infection comprising administering an effective amount of acompound of Formula (II)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereof.

In some embodiments, the present application includes pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acompound of Formula (I)

and/or a pharmaceutically acceptable salt and/or solvate thereofwhereinL is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN; andwherein the compound of Formula (I) is present in an amount effective totreat a nematode infection in a subject in need thereof.

In some embodiments, the present application includes pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acompound of Formula (II)

and/or a pharmaceutically acceptable salt and/or solvate thereof,wherein the compound of Formula (II) is present in an amount effectiveto treat a nematode infection in a subject in need thereof.

In some embodiments, the present application includes uses ofpharmaceutical compositions comprising a compound of Formula (I) and/or(II) for treating or preventing a nematode infection or a disease, adisorder, or a condition arising from a nematode infection in a subjectin need thereof.

In some embodiments, the present application includes nematicidalcompositions comprising a carrier and a compound of Formula (I)

and/or a salt and/or solvate thereofwhereinL is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN; andwherein the compound of Formula (I) is present in an amount effective totreat a nematode infection in a subject in need thereof.

In some embodiments, the present application includes nematicidalcompositions comprising a carrier and a compound of Formula (II)

and/or a salt, and/or solvate thereof wherein the compound of Formula(II) is present in an amount effective to treat a nematode infection ina subject in need thereof.

In some embodiments, the present application includes uses of thenematicidal composition described herein for treating or preventing anematode infection or a disease, a disorder, or a condition arising froma nematode infection in a subject in need thereof.

In some embodiments, the present application includes methods oftreating or preventing a nematode infection comprising administering aneffective amount of a nematicidal composition described herein to asubject in need thereof.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the application, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

DRAWINGS

The embodiments of the application will now be described in greaterdetail with reference to the attached drawings in which:

FIG. 1 shows movement defects induced by exemplary compounds includingcompound Ia. Panel A shows a negative control healthy C. elegans wormwithout drug. Panel B shows the “coiler defect” phenotype of a C.elegans worm in presence of exemplary Compound Ia.

FIG. 2 shows impact of exemplary compound Ia on HEK293 cellproliferation compared to a positive control where dashed line showsdata for exemplary compound Ia.

FIG. 3 shows drug-induced paralysis suppression in C. elegans in thepresence of exemplary compound Ia. Circle (●) indicates negative controlwhere no drug is used. Square (▪) shows positive control with Aldicarb,a paralysis-inducing drug. Downward-facing triangle (▾) indicatesexemplary compound Ia used in combination with Aldicarb. Upward-facingtriangle (▴) indicates exemplary compound Ia used alone.

DESCRIPTION OF VARIOUS EMBODIMENTS I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present application herein described for which theyare suitable as would be understood by a person skilled in the art.

In understanding the scope of the present application, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives.

The term “consisting” and its derivatives, as used herein, are intendedto be closed terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but exclude thepresence of other unstated features, elements, components, groups,integers and/or steps.

The term “consisting essentially of”, as used herein, is intended tospecify the presence of the stated features, elements, components,groups, integers, and/or steps as well as those that do not materiallyaffect the basic and novel characteristic(s) of features, elements,components, groups, integers, and/or steps.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

As used in this application, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.For example, an embodiment including “a compound” should be understoodto present certain aspects with compound or two or more additionalcompounds.

In embodiments comprising an “additional” or “second” component, such asan additional or second compound, the second component as used herein ischemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present. The term “and/or” with respect to pharmaceuticallyacceptable, salts and/or solvates thereof means that referencedcompounds exist as individual salts or hydrates, as well as acombination of, for example, a salt of a solvate of a compound.

The term “suitable” as used herein means that the selection of theparticular compound or conditions would depend on the specific syntheticmanipulation to be performed, the identity of the molecule(s) to betransformed and/or the specific use for the compound, but the selectionwould be well within the skill of a person trained in the art. Allprocess/method steps described herein are to be conducted underconditions sufficient to provide the product shown. A person skilled inthe art would understand that all reaction conditions, including, forexample, reaction solvent, reaction time, reaction temperature, reactionpressure, reactant ratio and whether or not the reaction should beperformed under an anhydrous or inert atmosphere, can be varied tooptimize the yield of the desired product and it is within their skillto do so.

The present description refers to a number of chemical terms andabbreviations used by those skilled in the art. Nevertheless,definitions of selected terms are provided for clarity and consistency.

The term “alkyl” as used herein, whether it is used alone or as part ofanother group, means straight or branched chain, saturated alkyl groups.The number of carbon atoms that are possible in the referenced alkylgroup are indicated by the prefix “C_(n1-n2)”. For example, the termC₁₋₁₀alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10carbon atoms.

The term “alkylene”, whether it is used alone or as part of anothergroup, means straight or branched chain, saturated alkylene group, thatis, a saturated carbon chain that contains substituents on two of itsends. The number of carbon atoms that are possible in the referencedalkylene group are indicated by the prefix “C_(n1-n2)”. For example, theterm C₁₋₆alkylene means an alkylene group having 1, 2, 3, 4, 5 or 6carbon atoms.

The term “cycloalkyl,” as used herein, whether it is used alone or aspart of another group, means a saturated carbocyclic group containingone or more rings. The number of carbon atoms that are possible in thereferenced cycloalkyl group are indicated by the numerical prefix“C_(n1-n2)”. For example, the term C₃₋₈cycloalkyl means a cycloalkylgroup having 3, 4, 5, 6, 7, or 8 carbon atoms.

The term “cycloalkenyl” as used herein, whether it is used alone or aspart of another group, means cyclic, unsaturated alkyl groups. Forexample, the term C₃₋₈cycloalkenyl means a cycloalkenyl group having 3,4, 5, 6, 7, or 8 carbon atoms and at least one double bond.

All cyclic groups, including phenyl, cycloalkyl and cycloalkenyl groups,contain one or more than one ring (i.e. are polycyclic). When a cyclicgroup contains more than one ring, the rings may be fused, bridged,spirofused or linked by a bond.

The term “benzofused” as used herein refers to a polycyclic group inwhich a benzene ring is fused with another ring.

A first ring being “fused” with a second ring means the first ring andthe second ring share two adjacent atoms there between.

A first ring being “bridged” with a second ring means the first ring andthe second ring share two non-adjacent atoms there between.

A first ring being “spirofused” with a second ring means the first ringand the second ring share one atom there between.

The term “substituted” as used herein means that the referenced atomcontains at least one substituent group other that a hydrogen atom.

The term “nematode infection” as used herein refers to an invasion ofcells or bodily tissues by a foreign undesirable nematode.

The term “anthelmintic” or “anthelmintics” as used herein refers to agroup of antiparasitic drug used in the treatment and prevention ofnematode infections.

As used herein, a compound with “anthelmintic activity” is a compound,which when tested, has measurable nematode-killing activity or resultsin sterility or reduced fertility in the nematodes such that fewerviable or no offspring result, or compromises the ability of thenematode to infect or reproduce in its host, or interferes with thegrowth or development of a nematode. The compound may also displaynematode repellant properties. The term “pharmaceutically acceptablesalt” means an acid addition salt or a basic addition salt suitable for,or compatible with, the treatment of subjects.

The term “pharmaceutically acceptable salts” embraces salts commonlyused to form addition salts of free acids or free bases. The nature ofthe salt is not critical, provided that it ispharmaceutically-acceptable. Suitable pharmaceutically acceptable acidaddition salts are prepared from an inorganic acid or an organic acid.Examples of such inorganic acids include, without limitation,hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. Examples of appropriate organic acids include, forexample, aliphatic, cycloaliphatic, aromatic, arylaliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include, without limitation, formic, acetic, propionic,succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid.Suitable pharmaceutically-acceptable base addition salts include, butare not limited to, metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, lysine and procaine.

The term “solvates” as used herein refers to complexes formed between acompound and a solvent from which the compound is precipitated or inwhich the compound is made. Accordingly, the term “solvate” as usedherein means a compound, or a salt a compound, wherein molecules of asuitable solvent are incorporated in the crystal lattice. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”.

The term “pharmaceutically acceptable solvate” means a solvate suitablefor, or compatible with, the treatment of subjects. For pharmaceuticallyacceptable solvates, a suitable solvent is physiologically tolerable atthe dosage used or administered.

The term “compound(s) of the application” as used herein refers to acompound of Formula (I) or Formula (II) and/or a pharmaceuticallyacceptable salt, and/or solvate thereof.

The expression “disease, disorder or condition arising from a nematodeinfection” as used herein refers to any disease, disorder or conditionthat is directly or indirectly caused by the presence of a nematodeinfection in a subject.

The term “subject” as used herein includes plants, seeds, soil, and allmembers of the animal kingdom including mammals and birds, and theirfood. Thus, the methods of the present application are applicable toplant treatment, human therapy and veterinary applications.

When used, for example, with respect to the methods of treatment, uses,compositions and kits of the application, a subject, for example asubject “in need thereof” is a subject who has been diagnosed with, issuspected of having, may come in to contact with, and/or was previouslytreated for a nematode infection or a disease, disorder or conditionarising from a nematode infection.

When used, for example, in respect to plant treatments, the compoundsand/or compositions may be delivered by several means includingpre-planting, post-planting and as a feed additive, drench, externalapplication, pill or by injection.

The term “pharmaceutical composition” as used herein refers to acomposition of matter for pharmaceutical use.

The term “pharmaceutically acceptable” means compatible with thetreatment of subjects.

The term “pharmaceutically acceptable carrier” means a non-toxicsolvent, dispersant, excipient, adjuvant or other material which ismixed with the active ingredient in order to permit the formation of apharmaceutical composition, i.e., a dosage form capable ofadministration to a subject.

The term “nematicidal composition” as used here in refers to acomposition of matter for managing one or more nematode infections.

The term “administered” as used herein means administration of aneffective amount of a compound, including compounds of Formula (I) or(II), or a salt and/or solvate thereof, to a cell either in cell cultureor in a subject.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and for periodsof time necessary to achieve a desired result. For example, in thecontext of treating a nematode infection, or a disease, disorder orcondition arising from a nematode infection, an effective amount of acompound is an amount that, for example, reduces the nematode infectioncompared to the nematode infection without administration of thecompound. By “reducing the infection”, it is meant, for example,reducing the amount of the infectious agent in the subject and/orreducing the symptoms of the infection. The amount of a given compoundor composition that will correspond to such an amount will varydepending upon various factors, such as the given compound orcomposition, the formulation, the route of administration, the type ofcondition, disease or disorder, the identity of the subject beingtreated, and the like, but can nevertheless be routinely determined byone skilled in the art.

The terms “to treat”, “treating” and “treatment” as used herein and asis well understood in the art, means an approach for obtainingbeneficial or desired results, including clinical results. Beneficial ordesired clinical results include, but are not limited to, diminishmentof extent of nematode infection, stabilization (i.e. not worsening) ofthe state of the nematode infection, preventing spread of the nematodeinfection, delay or slowing of infection progression, amelioration orpalliation of the nematode infectious state, diminishment of thereoccurrence of nematode infection, diminishment, stabilization,alleviation or amelioration of one or more diseases, disorders orconditions arising from the nematode infection, diminishment of thereoccurrence of one or more diseases, disorders or conditions arisingfrom the nematode infection, and remission of the nematode infectionand/or one or more symptoms or conditions arising from the nematodeinfection, whether partial or total, whether detectable or undetectable.“To treat”, “treating” and “treatment” can also mean prolonging survivalas compared to expected survival if not receiving treatment. “To treat”,“treating” and “treatment” as used herein also include prophylactictreatment. For example, a subject with an early nematode infection istreated to prevent progression, or alternatively a subject in remissionis treated to prevent recurrence.

“Palliating” an infection, disease, disorder and/or condition means thatthe extent and/or undesirable manifestations of an infection, disease,disorder and/or condition are lessened and/or time course of theprogression is slowed or lengthened, as compared to not treating theinfection, disease, disorder and/or condition.

The term “prevention” or “prophylaxis” and the like as used hereinrefers to a reduction in the risk or probability of a subject becomingafflicted with a nematode infection and/or a disease, disorder and/orcondition arising from a nematode infection or manifesting a symptomassociated with a nematode infection and/or a disease, disorder and/orcondition arising from a nematode infection.

II. Methods and Uses of the Application

Families of small molecule compounds have been identified thatincapacitate parasitic nematodes, and advantageously show no geneticresistance. This suggest that resistance to these compounds will be lesslikely to develop in the wild.

The present application includes methods and uses for treating orpreventing a nematode infection comprising administering an effectiveamount of a compound of Formula (I)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereofwhereinL is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN.

In one embodiment, the present application includes methods and uses fortreating or preventing a nematode infection comprising administering aneffective amount of a compound of Formula (II)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereof.

In some embodiments, the present application includes methods and usesof treating or preventing a disease, disorder or condition arising froma nematode infection comprising administering an effective amount acompound of Formula (I)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereofwhereinL is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN.

In some embodiments, the present application includes methods and usesof treating or preventing a disease, disorder or condition arising froma nematode infection comprising administering an effective amount of acompound of Formula (II)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereof.

In some embodiments, the present application includes uses ofpharmaceutical compositions comprising a compound of Formula (I) and/or(II) for treating or preventing a nematode infection or a disease, adisorder, or a condition arising from a nematode infection in a subjectin need thereof.

In some embodiments, the present application includes methods oftreating or preventing a nematode infection comprising administering aneffective amount of a nematicidal composition comprising a compound ofFormula (I) and/or (II) described herein to a subject in need thereof.

In some embodiments, the present application includes uses of anematicidal composition comprising a compound of Formula (I) and/or (II)described herein for treating or preventing a nematode infection or adisease, a disorder, or a condition arising from a nematode infection ina subject in need thereof.

In some embodiments, the L of compound of Formula (I) is CH₂.

In some embodiments, the L of compound of Formula (I) is absent, suchthat R¹ is directly bonded to the N atom.

In some embodiments, R¹ is C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each ofwhich is unsubstituted or substituted with a C₁₋₄alkyl group. In someembodiments, R¹ is C₃₋₈cycloalkyl which is unsubstituted. In someembodiments, R¹ is C₃₋₈cycloalkyl which is substituted with a C₁₋₄alkylgroup. In some embodiments, R¹ is C₃₋₈cycloalkenyl which isunsubstituted.

In some embodiments R¹ is unsubstituted C₅₋₇cycloalkyl.

In some embodiments, R² is H, CN or NO₂. In some embodiments, R¹ is CNor NO₂. In some embodiments, R² is CN. In some embodiments, R² is NO₂.In some embodiments, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the compound of Formula (I) is

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the nematode infection is an infection of anematode of a species selected from Cooperia oncophora, Haemonchuscontortus, and Caenorhabditis elegans.

Treatment methods comprise administering to a subject one or morecompounds of the application, and optionally consists of a singleadministration, or alternatively comprises a series of administrations.The length of the treatment period depends on a variety of factors, suchas the severity of the infection, disease, disorder or condition, theage of the subject, the dosage of the one or more compounds of theapplication, the activity of one or more compounds of the application,or a combination thereof.

In an embodiment, the one or more compounds of the application areadministered or used as soon as possible after exposure to the nematode.In an embodiment, the one or more compounds of the application areadministered or used until treatment of the nematode infection, diseasedisorder or condition is achieved. For example, until completeelimination of the nematode is achieved, or until the number of nematodehas been reduced to the point where the subject's defenses are no longeroverwhelmed and can kill any remaining nematode.

In an embodiment, the methods of the present application compriseadministering an effective amount of a compound or a composition of theapplication to a subject selected from humans, mammals, birds,vertebrates, plants, seeds, and soil.

In an embodiment, the uses of the present application of a compound or acomposition of the application are in a subject selected from humans,mammals, birds, vertebrates, plants, seeds, and soil.

In some embodiments, the nematode infects plants and the nematicidalcomposition is administered to the soil or to plants. In someembodiments, the nematicidal composition is administered to soil beforeplanting. In some embodiments, the nematicidal composition isadministered to soil after planting. In some embodiments, thenematicidal composition is administered to soil using a drip system. Insome embodiments, the nematicidal composition is administered to soilusing a drench system. In some embodiments, the nematicidal compositionis administered to plant roots or plant foliage (e.g., leaves, stems).In some embodiments the nematicide composition is tilled into the soilor administered in furrow. In some embodiments, the nematicidalcomposition is administered to seeds.

In some embodiments, the nematode parasite infects a vertebrate. In someembodiments, the nematicidal composition is administered to non-humanvertebrate. In some embodiments, the nematicidal composition isadministered to a human. In some embodiments, the nematicidalcomposition is formulated as a drench to be administered to a non-humananimal. In some embodiments, the nematicidal composition is formulatedas an orally administered drug. In some embodiments, the nematicidalcomposition is formulated as an injectable drug. In some embodiments,the nematicidal composition is formulated for topical applications suchas pour-ons, or for the use in tags or collars.

In some embodiments, the methods of the application compriseadministering a compound or a composition of the application through oneor more means selected from pre-planting, post-planting, as a feedadditive, a drench, an external application, a pill and by injection.

In some embodiments, the present application includes methods ofreducing the viability or fecundity or slowing the growth or developmentor inhibiting the infectivity of a nematode using a compound or acomposition of the application as described herein.

In some embodiments, the present application includes methods ofreducing the viability or fecundity or slowing the growth or developmentor inhibiting the infectivity of a nematode using a compound or acomposition of the application as described herein, the methodscomprising administering a compound or a composition of the applicationto subject selected from a human, a mammal, a bird, a vertebrate ingeneral, a plant, a seed, or soil. In some examples, the bird can be adomesticated fowl; the mammal can be a domesticated animal and/orlivestock.

The dosage of the one or more compounds of the application, variesdepending on many factors such as the pharmacodynamic propertiesthereof, the mode of administration, the age, health and weight/mass ofthe subject, the nature and extent of the symptoms, the frequency of thetreatment and the type of concurrent treatment, if any, and theclearance rate in the subject to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors. The one ormore compounds of the application may be administered initially in asuitable dosage that may be adjusted as required, depending on theresponse.

Compounds can be tested for anthelmintic activity using methods known inthe art. For example, the compound is combined with nematodes, e.g., ina well of microtiter dish, in liquid or solid media or in the soilcontaining the agent. Staged nematodes are placed on the media. The timeof survival, viability of offspring, and/or the movement of thenematodes are measured. An agent with “anthelmintic or anthelminthic orantihelmthic activity” can, for example, reduce the survival time ofadult nematodes relative to unexposed similarly staged adults, e.g., byabout 20%, 40%, 60%, 80%, or more. In the alternative, an agent with“anthelmintic or anthelminthic or antihelminthic activity” may alsocause the nematodes to cease replicating, regenerating, and/or producingviable progeny, e.g., by about 20%, 40%, 60%, 80%, or more. The effectmay be apparent immediately or in successive generations.

III. Compositions of the Application

In some embodiments, the present application includes pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acompound of Formula (I)

and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereofwherein:L is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN; andwherein the compound of Formula (I) is present in an amount effective totreat a nematode infection in a subject in need thereof.

In some embodiments, the present application includes pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acompound of Formula (I)I

and/or a pharmaceutically acceptable salt and/or solvate thereof,wherein the compound of Formula (I)I is present in an amount effectiveto treat a nematode infection in a subject in need thereof.

In some embodiments, the present application includes uses ofpharmaceutical compositions comprising a compound of Formula (I) or (II)for treating or preventing a nematode infection or a disease, adisorder, or a condition arising from a nematode infection in a subjectin need thereof.

In some embodiments, the present application includes a nematicidalcomposition comprising a carrier and a compound of Formula (I)

and/or a salt and/or solvate thereofwherein:L is C₀₋₄ alkylene;R¹ is phenyl, C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each of which isunsubstituted or substituted with one or two substituents independentlyselected from C₁₋₄alkyl, C₁₋₄fluoroalkyl and C₁₋₄alkyl substituted withCl or CN; andR² is H, NO₂, Cl, F or CN; andwherein the compound of Formula (I) is present in an amount effective totreat a nematode infection in a subject in need thereof.

In some embodiments, the present application includes a nematicidalcomposition comprising a carrier and a compound of Formula (II)

and/or a salt and/or solvate thereof wherein the compound of Formula(II) is present in an amount effective to treat a nematode infection ina subject in need thereof.

In some embodiments, the linker L of compound of Formula (I) is CH₂.

In some embodiments, the linker L of compound of Formula (I) is optionaland absent, such that R¹ is directly bonded to the N atom.

In some embodiments, R¹ is C₃₋₈cycloalkyl or C₃₋₈cycloalkenyl, each ofwhich is unsubstituted or substituted with a C₁₋₄alkyl group. In someembodiments, R¹ is C₃₋₈cycloalkyl which is unsubstituted. In someembodiments, R¹ is C₃₋₈cycloalkyl which is substituted with a C₁₋₄alkylgroup. In some embodiments, R¹ is C₃₋₈cycloalkenyl which isunsubstituted.

In some embodiments R¹ is unsubstituted C₅₋₇cycloalkyl.

In some embodiments, R² is H, CN or NO₂. In some embodiments, R¹ is CNor NO₂. In some embodiments, R² is CN. In some embodiments, R² is NO₂.

In some embodiments, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the compound of Formula (I) is

or a pharmaceutically acceptable salt and/or solvate thereof.

In some embodiments, the nematode infection is an infection of anematode of the following non-limiting, exemplary genera:Caenorhabditis, Nippostrongyles, Anguina, Ditylenchus, Tylenchorhynchus,Pratylenchus, Radopholus, Hirschmanniella, Nacobbus, Hoplolaimus,Scutellonema, Rotylenchus, Helicotylenchus, Rotylenchulus, Belonolaimus,Heterodera, other cyst nematodes, Meloidogyne, Criconemoides,Hemicycliophora, Paratylenchus, Tylenchulus, Aphelenchoides,Bursaphelenchus, Rhadinaphelenchus, Longidorus, Xiphinema, Trichodorus,Paratrichodorus, Dirofiliaria, Onchocerca, Brugia, Acanthocheilonema,Aelurostrongylus, Anchlostoma, Angiostrongylus, Ascaris, Bunostomum,Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme,Dipetalonema, Dracunculus, Enterobius, Filaroides, Haemonchus,Lagochilascaris, Loa, Manseonella, Muellerius, Necator, Nematodirus,Oesophagostomum, Ostertagia, Parafilaria, Parascaris, Physaloptera,Protostrongylus, Setaria, Spirocerca, Stephanogilaria, Strongy hides,Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella,Trichostrongylus, Trichuris, Uncinaria or Wuchereria. In someembodiments, the nematodes are of the genera Cooperia, Haemonchus,Caenorhabditis, Nippostrongyles, Dirofilaria, Onchocerca, Brugia,Acanthocheilonema, Dipetalonema, Loa, Mansonella, Parafilaria, Setaria,Stephanofilaria, Wucheria, Pratylenchus, Heterodera, Meloidogyne orParatylenchus. In some embodiments the nemotodes are of the speciesCooperia oncophora, Haemonchus contortus, Caenorhabditis elegans,Nippostrongyles brasiliensis, Ancylostoma caninum, Haemonchus contortus,Trichinella spiralis, Trichurs muris, Dirofilaria immitis, Dirofilariatenuis, Dirofilaria repens, Dirofilari ursi, Ascaris suum, Toxocaracanis, Toxocara cati, Strongyloides ratti, Parastrongyloides trichosuri,Heterodera glycines, Globodera pallida, Meloidogyne javanica,Meloidogyne incognita, Meloidogyne arenaria, Radopholus similis,Longidorus elongatus, Meloidogyne hapla or Pratylenchus penetrans.

In some embodiments, the nematode infection is an infection of anematode of a species selected from Cooperia oncophora, Haemonchuscontortus, Dirofilaria, Nippostrongyles brasiliensis and Caenorhabditiselegans.

In some embodiments, the subject is selected from humans, mammals,birds, vertebrates, plants, seeds, and soil.

In some embodiments, the nematicidal compositions further comprise oneor more agricultural excipients.

In some embodiments, the nematicidal compositions further comprise oneor more agriculturally acceptable excipients.

For example, in some embodiments, the nematicidal compositions furthercomprises an aqueous surfactant. Examples of surfactants that can beused include, Span 20, Span 40, Span 80, Span 85, Tween 20, Tween 40,Tween 80, Tween 85, Triton X 100, Makon 10, Igepal CO 630, Brij 35, Brij97, Tergitol TMN 6, Dowfax 3B2, Physan and Toximul TA 15, and mixturesthereof.

In some embodiments, the nematicidal composition further comprises apermeation enhancer (e.g., cyclodextrin). In some embodiments, thenematicidal composition further comprises a co-solvent. Examples ofco-solvents that can be used include ethyl lactate, methyl soyate/ethyllactate co-solvent blends (e.g., Steposol), isopropanol, acetone,1,2-propanediol, n-alkylpyrrolidones (e.g., the Agsolex series), apetroleum based-oil (e.g., aromatic 200) or a mineral oil (e.g.,paraffin oil), or mixtures thereof. In some embodiments, the nematicidalcomposition further comprises another pesticide (e.g., nematicide,insecticide or fungicide) such as an avermectin (e.g., ivermectin),milbemycin, imidacloprid, aldicarb, oxamyl, fenamiphos, fosthiazate,metam sodium, etridiazole, penta-chloro-nitrobenzene (PCNB), flutolanil,metalaxyl, mefonoxam, and fosetyl-al, or mixtures thereof. Usefulfungicides include, but are not limited to, silthiofam, fludioxonil,myclobutanil, azoxystrobin, chlorothalonil, propiconazole, tebuconazoleand pyraclostrobin, or mixtures thereof. In some embodiments, thenematicidal composition may also comprise herbicides (e.g.,trifloxysulfuron, glyphosate, halosulfuron) and other chemicals fordisease control (e.g., chitosan).

In some embodiments, the application also includes a nematicidal feedfor a non-human vertebrate including:

(a) a feed; and (b) a nematicidal composition, of the application.

In some embodiments, the feed is selected from: soy, wheat, corn,sorghum, millet, alfalfa, clover, and rye, and mixtures thereof. Alsodescribed are feeds that have been supplemented to include one or moreof the compounds of the application. A nematicidal feed for a non-humanvertebrate can comprise: (a) an animal feed; and (b) an effective amountof a nematicidal compound of the application.

In some embodiments, the pharmaceutical compositions further compriseone or more pharmaceutically acceptable excipients. Conventionalprocedures and ingredients for the selection and preparation of suitablepharmaceutical compositions are described, for example, in Remington'sPharmaceutical Sciences (2000-20th edition) and in The United StatesPharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

For example, the pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form is sterile and fluid to the extentthat easy syringability exists.

In an embodiment, parenteral administration is by continuous infusionover a selected period of time. Solutions suitable for parenteraladministration are prepared by known methods by a person skilled in theart. For example, the compounds of the application are prepared in wateroptionally mixed with a surfactant such as hydroxypropylcellulose.Dispersions are also prepared in glycerol, liquid polyethylene glycols,DMSO and mixtures thereof with or without alcohol, and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

Compositions for nasal administration are conveniently formulated asaerosols, drops, gels or powders. Aerosol formulations typicallycomprise a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which take the form of a cartridge or refill for usewith an atomising device. Alternatively, the sealed container is aunitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve, which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it contains a propellant, which is, for example, a compressedgas such as compressed air or an organic propellant such asfluorochlorohydrocarbon. In an embodiment, the aerosol dosage forms takethe form of a pump-atomizer.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, wherein the active ingredient isformulated with a carrier such as sugar, acacia, tragacanth, gelatinand/or glycerine. Compositions for rectal administration areconveniently in the form of suppositories containing a conventionalsuppository base such as cocoa butter.

In another embodiment, compounds of the application are orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they are enclosed in hard or soft shell gelatincapsules, or they are compressed into tablets, or they are incorporateddirectly with the food of a diet. For oral administration, the compoundsof the application may be incorporated with excipients and used in theform of, for example, ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Oraldosage forms also include modified release, for example immediaterelease and timed-release, formulations. Examples of modified-releaseformulations include, for example, sustained-release (SR),extended-release (ER, XR, or XL), time-release or timed-release,controlled-release (CR), or continuous-release (CR or Contin), employed,for example, in the form of a coated tablet, an osmotic delivery device,a coated capsule, a microencapsulated microsphere, an agglomeratedparticle, e.g., molecular sieving type particles, or, a fine hollowpermeable fiber bundle, or chopped hollow permeable fibers, agglomeratedor held in a fibrous packet. In an embodiment, timed-releasecompositions are, formulated, as liposomes or those wherein the activecompound is protected with differentially degradable coatings, such asby microencapsulation, multiple coatings, etc. Liposome delivery systemsinclude, for example, small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. In an embodiment, liposomes areformed from a variety of lipids, such as cholesterol, stearylamine orphosphatidylcholines.

It is also possible to freeze-dry the compounds of the application anduse the lyophilizate obtained, for example, for the preparation ofproducts for injection.

The compounds of the application are either commercially available ormay be prepared using methods known in the art.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The formation of solvates will vary depending on the compound and thesolvate. In general, solvates are formed by dissolving the compound inthe appropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions. The selection of suitable conditions to form a particularsolvate can be made by a person skilled in the art.

The following non-limiting examples are illustrative of the presentapplication. As is apparent to those skilled in the art, many of thedetails of the examples may be changed while still practicing themethods, compositions and kits described herein.

IV. Methods of Preparation

The compounds of the application are either commercially available ormay be prepared using methods known in the art. For example, in someembodiments, compounds of Formula (I) are prepared as shown in Scheme I:

Therefore compounds of Formula (I), wherein R² is as defined in Formula(I), are reacted with excess amounts of piperazine (B) in the presenceof an inorganic base at elevated temperatures to provide compounds ofFormula C. Compounds of Formula C are then reacted with compounds ofFormula D, wherein LG is a leaving group and R¹ is as defined in Formula(I), in the presence of an organic base, at elevated temperatures, toprovide compounds of Formula (I).

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid ina suitable solvent and the formed salt is isolated by filtration,extraction or any other suitable method.

The formation of solvates will vary depending on the compound and thesolvate. In general, solvates are formed by dissolving the compound inthe appropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions. The selection of suitable conditions to form a particularsolvate can be made by a person skilled in the art.

The following non-limiting examples are illustrative of the presentapplication. As is apparent to those skilled in the art, many of thedetails of the examples may be changed while still practicing themethods, compositions and kits described herein.

EXAMPLES Example 1: Preparation of Compounds Materials and Methods

Unless otherwise stated, all reactions were carried out under anatmosphere of dry argon, using glassware that was either oven (120° C.)or flame-dried. Work-up and isolation of compounds was performed usingstandard benchtop techniques. All commercial reagents were purchasedfrom chemical suppliers (Sigma-Aldrich, Combi-Blocks, or Alfa Aesar) andused without further purification. Dry solvents were obtained usingstandard procedures (dichloromethane and acetonitrile were distilledover calcium hydride). Reactions were monitored using thin-layerchromatography (TLC) on EMD Silica Gel 60 F254 plates. Visualization wasperformed under UV light (254 nm) or using potassium permanganate(KMnO₄) stain. Flash column chromatography was performed on SiliaflashP60 40-63 μm silica gel purchased from Silicycle.

NMR characterization data was obtained at 293K on a Varian Mercury 300MHz, Varian Mercury 400 MHz, Bruker Advance III 400 MHz, Agilent DD2 500MHz equipped with a 5 mm Xses cold probe or Agilent DD2 600 MHz. ¹Hspectra were referenced to the residual solvent signal (CDCl₃=7.26 ppm,DMSO-d₆=2.50 ppm). ¹³C{¹H} spectra were referenced to the residualsolvent signal (CDCl₃=77.16 ppm, DMSO-d₆=39.52 ppm). Data for ¹H NMR arereported as follows: chemical shift (δ ppm), multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet), coupling constant (Hz),integration. NMR spectra were recorded at the University of TorontoDepartment of Chemistry NMR facility(http://www.chem.utoronto.ca/facilities/nmr/nmr.html).

Infrared spectra were recorded on a Perkin-Elmer Spectrum 100 instrumentequipped with a single-bounce diamond/ZnSe ATR accessory in the solidstate and are reported in wavenumber (cm⁻¹) units.

High resolution mass spectra (HRMS) were recorded at the AdvancedInstrumentation for Molecular Structure (AIMS) in the Department ofChemistry at the University of Toronto(https://www.chem.utoronto.ca/chemistry/AIMS.php).

High-performance liquid chromatography (HPLC) analyses were performed onan Agilent 1100 series instrument containing a Phenomenex XDB-C18 column(1.8 μm, 50×4.6 mm) and run at room temperature. A linear gradientstarting from 5% acetonitrile and 95% water (0.1% formic acid) to 95%acetonitrile and 5% water (0.1% formic acid) over 4 minutes followed byelution for 5 minutes at 95% acetonitrile and 5% water (0.1% formicacid) was used. The flow rate was set to 1.0 mL/min, with UV detectionat 254 and 280 nm.

General Procedure A

The procedure was modified from literature (Sokoloff, P.; Pilon, C.;Mann, A.; Schoenfelder, A.; Garrido, F. 3,4-dihydro-2-naphthamidederivatives as selective dopamine D3 ligands. European PatentApplication 05290156.8, Jul. 26 2006). To a round-bottom flask at roomtemperature were added piperazine (1.38 g, 16 mmol, 4.0 equiv),potassium carbonate (1.11 g, 8 mmol, 2 equiv), dimethyl sulfoxide (4.7mL) then the corresponding fluorobenzene derivative (4 mmol, 1 equiv)and the mixture was stirred at 100° C. Once the reaction was complete asindicated by TLC (approximately 22 h), the reaction mixture was dilutedwith ethyl acetate, transferred to a separatory funnel and washed withwater (3 times) then saturated sodium chloride. The organic phase wasdried with Na₂SO₄, filtered, then concentrated on a rotary evaporatorand the substituted 1-phenylpiperazine (C) was obtained. The crude solidwas used in the next step without further purification.

General Procedure B

To a round-bottom flask were added C (0.5 mmol, 1.0 equiv), dryacetonitrile (1 mL), triethylamine (0.14 mL, 1 mmol, 2 equiv) then thealkyl halide wherein X is a halide (D, 0.5 mmol, 1.0 equiv) and themixture was stirred at reflux until completion as indicated by TLC(approximately 24 h). The mixture was diluted with ethyl acetate,transferred to a separatory funnel and washed with water (3 times) thensaturated sodium chloride. The organic phases were combined and driedwith Na₂SO₄, filtered, and concentrated on a rotary evaporator. Theresidue was purified by column chromatography with the indicated eluentand the alkylated arylpiperazines (I) were obtained.

Preparation of cyclopentylmethyl 4-methylbenzenesulfonate

The procedure was modified from literature (Shi, W.; Nacev, B. A.;Aftab, B. T.; Head, S.; Rudin, C. M.; Liu, J. O. J. Med. Chem. 2011, 54,7363). To a round-bottom flask were added cyclopentylmethanol (0.087 mL,0.8 mmol, 1.0 equiv) and dry dichloromethane (1.6 mL) and the flask wassubmerged in an ice-water bath. Triethylamine (0.17 mL, 1.2 mmol, 1.5equiv) was added to the flask followed by 4-(dimethylamino)pyridine(48.9 mg, 0.4 mmol, 0.5 equiv) then p-toluenesulfonyl chloride (168 mg,0.88 mmol, 1.1 equiv) and the mixture was warmed to room temperature andstirred until completion as indicated by TLC (approximately 3 h).Dichloromethane and water were added, then the mixture was transferredto a separatory funnel and the layers were separated. The organic phasewas washed with water (2 times) followed by saturated sodium chloride,then dried over Na₂SO₄, filtered, and concentrated on a rotaryevaporator. The residue was purified by column chromatography (19/1 v/vpentanes/ethyl acetate) and cyclopentylmethyl 4-methylbenzenesulfonate(D(a), 59% yield) was obtained as a colourless oil. The spectral datawas in accordance with literature.

Example 2 Screening of Compounds

Because of their dependence on a host, parasitic nematodes are difficultto culture, maintain and sustain. Therefore, to screen forantithelmintic compounds, the non-parasitic nematode, C. elegans, wasused as a model system, then compound leads were investigated inparasitic nematodes.

Molecules that can modulate the egg-laying rate of the C. elegans (whichcan be done in medium-throughput fashion) were identified first and thenthe hits' ability to disrupt worm locomotion was examined usinglower-throughput techniques. Egg-laying is a convenient system withwhich to interrogate neuromuscular control because it's digital output(an egg is either laid or not) can be easily quantified. Wild typehermaphrodites carry 10-15 eggs in their uterus and lay ˜one egg perhour in the liquid (control) buffer used for liquid worm culture.Egg-laying is principally regulated by cholinergic and serotonergicsignaling via conserved receptors.

To identify egg-laying (Egl) modulators, a medium-throughputline-scanning microscope capable of imaging a 96 well plate in just over2 minutes was used. From the resulting images, image analysis softwareto count the number of eggs laid per adult per hour in each well wasused. Small molecules at a final concentration of 60 μM were screened,which is a technically convenient concentration where most moleculesstay in solution. This may be considered a high concentration for mostscreening platforms, but is suitable for C. elegans because of thenematode's robust xenobiotic defenses.

A collection of 486 synthetic small molecules that have previously shownsome effect on worm growth was screened. These molecules are fromChembridge Inc., are drug-like in their physico-chemical properties, andhave relatively uncharacterized bioactivity. To identifyEgl-stimulators, small molecules were screened in a benign controlbuffer. To identify Egl-inhibitors, molecules were screened in thebackground of 2.5 mM serotonin plus 7.7 mM nicotine, which is a cocktailthat was empirically determined to induce robust egg-laying in liquidmedia. Molecules that reproducibly (p<0.05) modulate egg-laying two foldgreater than control conditions were considered ‘stimulators’, andmolecules that modulate egg-laying two fold less than stimulatingconditions were considered ‘inhibitors’.

In the above-described assay, 31 stimulators and 33 inhibitors ofegg-laying were identified. A structural comparison of these hits usinga Tanimoto™/FP2 similarity cutoff of 0.55 or greater revealed that manyof the Egl-modulators belong to structurally-similar families. A networkwas generated using a Tanimoto™ chemical similarity cut-off of 55%.Molecules that share structural similarity typically modulate egg layingsimilarly, suggesting a shared mechanism of action.

The ability of the 64 Egl hits to modulate the normal sinusoidallocomotion of wild type C. elegans was then examined. The animal'sbehaviour over the course of several hours was manually monitored whilethe animal moved atop a solid agar substrate containing the smallmolecule. Many different genes govern the sinusoidal locomotion of wildtype C. elegans, but their mutation can lead to qualitatively distinctlocomotory phenotypes. Hence, a distinct locomotory response to a smallmolecule can be suggestive of its mechanism of action.

Compared to the normal phenotype of the worm as shown in FIG. 1 panel A,8 molecules that induce simultaneous anterior and posterior contraction(called a ‘rubber-band’ phenotype) were identified; 3 molecules thatcause animals to turn along their circumferential axis (ca a ‘shaker’phenotype) were identified; 3 molecules that cause the animal to coillike a snake (called a ‘coiler’ phenotype) were identified; 2 moleculesthat induce jerky uncoordination (called jerky-unc) were identified; 3molecules that disrupt reverse locomotion were identified; and 3 thatinduce paralysis were identified.

Compound Ia induced a strong coiler phenotype (FIG. 1 panel B). Previousphylogenetic bioactivity analysis suggests that the activity of compoundIa might be restricted to nematodes. The coiler phenotype that isinduced by compound Ia is consistent with a decrease in acetylcholine(ACh) levels at the synaptic cleft.

Example 3 Cytotoxicity of Exemplary Compound Ia

The cytotoxicity of exemplary compound Ia was investigated by measuringHEK293 cell proliferation in a dose-response analysis and no effect wasfound on HEK293 cell proliferation by exemplary compound Ia as shown inFIG. 2. A cytotoxic formulation was used as positive control.Experiments were performed in triplicates on different days. Cellviability was assess using a standard Celltiter Blue™ Cell ViabilityAssay (Promega). Compounds were added to cells in 96-well plates at timezero and cell viability assessed in 48 hours. Error bars indicatestandard deviation.

Example 4 Compounds of Formula (I) Induce Coiler Phenotype

Three structurally-related molecules (compound Ia, compound Ie andcompounds Ic) induce a ‘severe’ snake-like coiler phenotype andparalysis (FIG. 1, panel B). Compound Id is a forth structurally-relatedmolecule that induces a ‘jerky-unc’ phenotype. A detailed phenotypicanalyses suggests that the animals exposed to these moleculesphenotypically transit over time from jerky-unc to being coiled to beingparalyzed, with exemplary compound Ia being the prototypical example.

Forward genetic screens were performed for mutants that can resist theearly larval lethal effects of compound Ic and compound Id. Over 200,000F1 animals and over 100,000 F2 animals from randomly mutagenizedparents/grandparents were screened. No mutants that could resist thelethal effects of compound Ic and compound Id. This suggests that viableresistance may be difficult to achieve against this class of nematicidein the wild.

Example 5 Mechanism of Action: Compounds of Formula (I) Likely InhibitsVesicular Acetylcholine Transporter (VAChT)

Two observations raised the possibility that compounds of Formula (I)reduce acetylcholine (ACh) signalling. First, many mutant genes thatyield a strong coiler phenotype are associated with disruptedcholinergic motorneuron development and/or with disrupted AChproduction, release or reception. Second, the compounds of Formula (I)have structural similarity to vesamicol, which is a research tool thatinhibits the vertebrate vesicular acetylcholine transporter (VAChT)(Table 1). VAChT transports cytoplasmic ACh into synaptic vesicles inthe presynaptic membrane of all animals. Vesamicol is not useful as amedicine because it disrupts ACh signalling and is therefore toxic tovertebrates. In C. elegans, VAChT is encoded by unc-17, whosereduction-of-function phenotype is ‘severe’ coiling. It was thereforehypothesized that exemplary compound Ia inhibits UNC-17.

Exemplary compound Ia's inhibitory activity on UNC-17 was tested inseveral ways. First, the phenotype of exemplary compound Ia was comparedto that of vesamicol. Vesamicol induces jerky-unc and coiling, but onlyat concentrations close to its limit of solubility on solid substrate.This analysis reveals that exemplary compound Ia is more than 30 timesmore potent than vesamicol for inducing locomotory defects in worms.

Whether exemplary compound Ia can suppress the paralysis that is inducedby aldicarb was tested. Aldicarb inhibits acetylcholinesterase, whichnormally catabolizes ACh at the synaptic cleft. Exemplary compound Ia'sEC₅₀ for suppression of Aldicarb activity is 4.806 μM. Aldicarbtreatment therefore results in excess ACh and paralyzes the animal dueto excess muscle contraction. If exemplary compound Ia inhibits UNC-17and results in lower levels of ACh at the synaptic cleft, then exemplarycompound Ia should reduce aldicarb-induced paralysis. Indeed, an obviousreduction of aldicarb-induced paralysis by exemplary compound Ia wasfound (p<0.001) (FIG. 3).

Third, the interaction between exemplary compound Ia and various mutantstrains of unc-17 was explored. If exemplary compound Ia inhibitsUNC-17, then unc-17 reduction-of-function alleles should behypersensitive to exemplary compound Ia. Indeed, the unc-17 hypomorphicalleles e245, e327, and e795 have heightened sensitivity to exemplarycompound Ia (p<0.001). Previous work demonstrated that the C391Ymutation in human VAChT is essential for vesamicol binding. Theorthologous mutation in UNC-17 is C370Y and exists in the unc-17(md414)mutant, which also has weak reduction-of-function phenotypes. Withoutknowing that human VAChT (C391Y) fails to interact with vesamicol, thenaïve expectation is that unc-17(md414) would also be hypersensitive tocompound Ia. However, it was found that unc-17(md414) is partiallyresistant to the effects of both compound Ia and vesamicol (p=0.001).Together, these chemical-genetic interactions indicate that compound Ialikely inhibits UNC-17 function.

Example 6 Bioactivity Profile of Exemplary Compound Ia

Compounds of the Formula (I) fully inhibit egg-hatching of the mammalianparasites H. contortus and C. oncophora at mid-micromolar concentrations(30-60 uM) and inhibit dog heartworm movement with an ED₅₀ in the lowmicromolar concentration range (2-10 uM) in ex-vivo assays. Thecompounds have little effect on HEK293 cell proliferation at anyconcentration tested as shown above in FIG. 2.

A dose-response analysis in fish of three compounds of Formula (I)showed that the molecules induce cardiac defects in the low micromolarrange. However, exemplary compound Ia itself has the weakest cardiacdefects of the three compounds, while compound Ie (which has relativelyweak effects in worms), induced the strongest cardiac defects.

Example 7 Structural-Activity Relationship (SAR) of Compounds of Formula(I) and Bioactivity Profile

A preliminary first round of SAR analyses using analogs of exemplarycompound Ia was performed and the analogs' ability to induce locomotorydefects in wild type fourth-stage C. elegans larvae (L4s) was tested. Asexpected, most analogs induced weaker locomotory phenotypes compared tocompound Ia, the exception being compound Ib (Table 1). Using thisinformation, new analogs of exemplary compound Ia were synthesizedand/or purchased, and the most potent analog to date, compound If(Table 1) was identified.

Table 1 shows the preliminary SAR study conducted on compounds ofFormula (I), where activity indicates the percent of animals that showcoiled and/or paralysis after 60 min on solid substrate with a “drug”concentration 60 μM. If phenotype is observed, a dose-response analysisis performed. Exemplary compound Ia has an EC₅₀ of 12.8 μM.

Example 8 Structural-Activity Relationship (SAR) of Compounds of Formula(I) and Bioactivity Profile

Table 2 shows the extension of the SAR study conducted on compounds ofFormula (I), where data are the % of L3 animals demonstrating abnormallocomotory behaviour on solid agar (MYOB medium+E. coli OP50 foodsource) containing the indicated compound of the Application at 3.75 uM,15 uM or 60 uM (as noted). 20 animals were scored after either 1, 6 or24 hours of exposure to drug (as indicated) per replicate. Abnormallocomotory phenotype (phene) is defined as animals demonstrating 1 ormore of the following phenotypes: jerky locomotion, flaccid paralysis,coiling. Data are reported as the mean % of animals with abnormalphenotype over 4 independent replicates.

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the present application is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present application is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

TABLE 1 Compound R² L—R¹ Activity (%) Ia NO₂

90% +/− 5.4 Ig F

11% +/− 5.6 Ih Cl

2% +/− 1.9 Ii H

19% +/− 8.1 Ik CN

61% +/− 5.0 Ib NO₂

87% +/− 3.7 Im NO₂

0.00 If NO₂

98% +/− 1.9 Ip CN

41% +/− 7.4 Iq NO₂

6% +/− 3.2 Ic NO₂

71% +/− 2.1 Id NO₂

0.00 Ie NO₂

19% +/− 8.0 Vesamicol — — 6% +/− 0.1 Piperazine — — 0.00

TABLE 2 % any phene 1 h (conc.) % any phene 6 h (conc.) % any phene 24 h(conc.) Compound I.D. 3.75 15 60 3.75 15 60 3.75 15 60 Control 0 0 0 0 00 0 0 0 Ia 42.5 91.0 100.0 22.5 64.2 100.0 0.0 34.2 80.0 Ic 29.2 95.9100.0 8.3 80.8 100.0 0.0 89.2 100.0 Id 19.2 65.5 95.8 5.8 36.7 100.0 0.045.0 95.8 Ie 15.0 45.0 94.2 0.0 56.7 80.8 0.0 0.0 57.5 Ib 3.3 97.5 100.00.0 93.3 100.0 0.0 73.3 100.0 Im 0.0 7.5 44.2 0.0 3.3 30.8 0.0 0.0 0.0Ig 21.7 44.2 95.0 0.8 9.2 79.2 0.0 4.2 66.7 Ih 0.0 27.5 55.7 0.0 0.0 5.80.0 0.0 10.8 Ii 16.2 39.2 84.2 0.0 0.8 44.2 0.0 0.0 20.5 Ir 43.3 97.5100.0 9.2 88.3 100.0 0.0 30.0 100.0 Is 3.3 17.5 100.0 0.0 15.0 62.5 0.00.0 13.3 Ik 8.3 45.8 91.7 15.0 37.5 98.3 0.0 23.3 100.0 Ip 0.8 29.2 91.30.0 10.0 42.5 0.0 0.0 62.5 Iq 0.0 4.2 32.5 5.0 29.2 36.7 0.0 0.0 4.2 Iw5.0 14.2 37.5 0.0 1.7 19.2 0.0 0.0 3.3 Ix 89.2 100.0 100.0 100.0 100.0100.0 66.7 100.0 100.0 Iy 22.5 77.5 100.0 0.0 35.0 99.2 0.0 0.0 58.3 Iz60.8 96.7 99.2 0.0 6.7 85.0 0.0 0.0 14.2 Iaa 45.0 89.2 100.0 6.7 27.597.5 0.0 0.0 69.2 Ibb 27.5 92.5 100.0 5.0 75.8 100.0 0.0 13.3 100.0 Icc46.7 93.3 100.0 0.0 0.8 33.3 0.0 0.0 0.0 Idd 92.5 100.0 100.0 86.7 100.097.5 34.2 98.3 97.5 Iff 24.2 98.3 100.0 6.7 28.3 51.7 0.0 0.0 0.0 Igg25.8 80.0 100.0 0.0 68.3 100.0 0.0 0.0 100.0 Ihh 4.2 70.0 100.0 0.0 1.753.3 0.0 0.0 5.0 Iii 81.7 96.7 100.0 14.2 94.2 100.0 0.0 77.5 100.0vesamicol 0.0 0.0 11.7 0.0 0.8 1.7 0.0 0.0 0.0

1. A method of treating or preventing a nematode infection comprisingadministering an effective amount of a compound of Formula (I) and/or apharmaceutically acceptable salt and/or solvate thereof, to a subject inneed thereof, wherein the compound of Formula (I) is selected from


2. The method of claim 1, wherein the compound of Formula (I) isselected from

or a pharmaceutically acceptable salt and/or solvate thereof.
 3. Themethod of claim 1, wherein the compound of Formula (I) is

or a pharmaceutically acceptable salt and/or solvate thereof.
 4. Themethod of claim 1, wherein the infection is an infection of a nematodeof a species selected from Cooperia oncophora, Haemonchus contortus, andCaenorhabditis elegans.
 5. The method claim 1, wherein the subject isselected from a human, a mammal, a bird, a plant, a seed, and soil. 6.The method of claim 1, wherein the subject is selected from a plant, aseed and soil and the compound is administered pre-planting,post-planting, as a feed additive, a drench, an external application, apill and/or by injection.
 7. A method of treating or preventing adisease, disorder or condition arising from a nematode infectioncomprising administering an effective amount a compound of Formula (I)and/or a pharmaceutically acceptable salt and/or solvate thereof to asubject in need thereof wherein the compound of Formula (I) is selectedfrom


8. The method of claim 7, wherein the compound of Formula (I) isselected from

or a pharmaceutically acceptable salt and/or solvate thereof.
 9. Themethod of claim 7, wherein the compound of Formula (I) is

or a pharmaceutically acceptable salt and/or solvate thereof.
 10. Themethod of claim 7, wherein the infection is an infection of a nematodeof a species selected from Cooperia oncophora, Haemonchus contortus, andCaenorhabditis elegans.
 11. The method claim 7, wherein the subject isselected from a human, a mammal, a bird, a plant, a seed, and soil. 12.The method of claim 7, wherein the subject is selected from a plant, aseed and soil and the compound is administered pre-planting,post-planting, as a feed additive, a drench, an external application, apill and/or by injection.
 13. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of Formula (I) and/ora pharmaceutically acceptable salt and/or solvate thereof Wherein thecompound of Formula (I) is selected from

and the compound of Formula (I) is present in an amount effective totreat a nematode infection in a subject in need thereof.
 14. Thepharmaceutical composition of claim 13, wherein the compound of Formula(I) is selected from

or a pharmaceutically acceptable salt and/or solvate thereof.
 15. Thepharmaceutical composition of claim 13, wherein the compound of Formula(I) is

or a pharmaceutically acceptable salt and/or solvate thereof.
 16. Amethod of treating or preventing a nematode infection comprisingadministering an effective amount of a composition claim 13 to a subjectin need thereof.
 17. The method of claim 16, wherein the subject isselected from a plant, a seed and soil and the composition isadministered pre-planting, post-planting, as a feed additive, a drench,an external application, a pill and by injection.